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Subjects

Abstract

Repair of tissue damaged during inflammatory processes is key to the return of local homeostasis and restoration of epithelial integrity. Here we describe CD161+ regulatory T (Treg) cells as a distinct, highly suppressive population of Treg cells that mediate wound healing. These Treg cells were enriched in intestinal lamina propria, particularly in Crohn’s disease. CD161+ Treg cells had an all-trans retinoic acid (ATRA)-regulated gene signature, and CD161 expression on Treg cells was induced by ATRA, which directly regulated the CD161 gene. CD161 was co-stimulatory, and ligation with the T cell antigen receptor induced cytokines that accelerated the wound healing of intestinal epithelial cells. We identified a transcription-factor network, including BACH2, RORγt, FOSL2, AP-1 and RUNX1, that controlled expression of the wound-healing program, and found a CD161+ Treg cell signature in Crohn’s disease mucosa associated with reduced inflammation. These findings identify CD161+ Treg cells as a population involved in controlling the balance between inflammation and epithelial barrier healing in the gut.

Acknowledgements

The authors thank patients who contributed samples toward this study. This work was supported by the Wellcome Trust (grant 097261/Z/11/Z to B.A. and WT101159 to N.P.), the Crohn’s and Colitis Foundation of America (grant CCFA no. 3765 — CCFA genetics initiative to A.L.), British Heart Foundation (grant RG/13/12/30395 to G.L.), institutional start-up fund from Purdue University and National Heart, Lung, and Blood Institute (grant 5K22HL125593-02 to M.K.). Research was also supported by the National Institute for Health Research (NIHR) Biomedical Research Centre at Guy’s and St Thomas’ National Health Service (NHS) Foundation Trust and King’s College London. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. This research was supported (in part) by the Intramural Research Programs of the National Institute of Arthritis and Musculoskeletal and Skin Diseases, the National Institute of Diabetes and Digestive and Kidney Diseases and the National Heart, Lung, and Blood Institute of the National Institutes of Health. The authors thank J. O’Shea (National Institutes of Health) for his support and for providing access to ATAC-seq, the National Heart, Lung, and Blood Institute DNA Sequencing and Genomics Core for performing single-cell sequencing experiment and acknowledge the assistance of M. Arno (Genomics Centre, King’s College London) with gene expression microarray studies as well as S. Heck and R. Ellis (Biomedical Research Centre Flow Core Facility, King’s College London) for CyTOF data acquisition. In addition, the authors thank E. Mathé (Ohio State University) for critically reading the manuscript.

Author information

Author notes

These authors contributed equally: Giovanna Lombardi, Behdad Afzali.

Affiliations

MRC, Centre for Transplantation, King’s College London, London, UK

Giovanni A. M. Povoleri

, Estefania Nova-Lamperti

, Cristiano Scottà

, Giorgia Fanelli

, Pablo D. Becker

, Dominic Boardman

, Marco Romano

, Polychronis Pavlidis

, Reuben McGregor

, Eirini Pantazi

, Nick Powell

, Giovanna Lombardi

& Behdad Afzali

National Institute for Health Research Biomedical Research Centre at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London, London, UK

Corresponding author

Integrated supplementary information

(a) Heat map showing signal intensity of selected markers for each node of the populations shown in Fig. 1c. (b–e) Clustering of populations of Tconv and Treg cells by single-cell RNA-seq (scRNAseq) showing the tSNE plot of 2,636 cells from n= 3 independent donors separated into six major (clusters 0–6) and two minor (clusters 7 and 8) clusters (b), feature plots showing expression of three Treg markers (IL2RA encoding CD25, IL7R encoding CD127 and FOXP3 encoding FOXP3), KLRB1 (encoding CD161) and two naive/memory markers (SELL encoding CD62L and CCR7 encoding CCR7) (c), a heat map showing the top 20 discriminatory genes per cluster with representative genes of each cluster indicated (d) and population clustering (e) based on expression of the transcripts shown in c. Clusters in b are color-coded and labeled according to expression of the markers shown in e. (f) Clustering of Tconv and Treg subpopulations using protein expression data sourced from CyTOF in Fig. 1a incorporating the same markers as in e and also including CD45RA and CD45RO. Clustering of subpopulations in f is similar to that from scRNAseq (e). (g,h) viSNE plots (g) and 2D minimum spanning tree (h) of CD4+ T cells clustered following staining with anti-CD4, anti-CD25, anti-CD127, anti-CD45RA and anti-CD161 for flow cytometry. Shown in g are heat maps for expression of the indicated markers, with an arrow indicating CD161 expression in Treg cells. Node size in h represents cell number and color CD161 median intensity. Grouped together are naive (circled in orange), memory (circled in yellow) and CD161+ (circled in purple) Treg cells, as well as populations of naive (circled in black), memory (circled in black) and CD161+ (circled in red) Tconv cells. The data in g and h are representative of n = 4 experiments. *P < 0.01, **P < 0.001, ***P < 0.0001 by one-way ANOVA.